help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 1, 2002, doi:10.1164/rccm.200204-285OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200204-285OCv1
166/11/1498    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reynolds, S. D.
Right arrow Articles by Stripp, B. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reynolds, S. D.
Right arrow Articles by Stripp, B. R.
American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1498-1509, (2002)
© 2002 American Thoracic Society

Original Article

Secretoglobins SCGB3A1 and SCGB3A2 Define Secretory Cell Subsets in Mouse and Human Airways

Susan D. Reynolds, Paul R. Reynolds, Gloria S. Pryhuber, Jonathan D. Finder and Barry R. Stripp

Departments of Environmental and Occupational Health, and Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania; and Department of Pediatrics, University of Rochester, Rochester, New York

Correspondence and requests for reprints should be addressed to Susan D. Reynolds, Ph.D., Department of Environmental and Occupational Health, Room 304, 3343 Forbes Avenue, University of Pittsburgh, Pittsburgh, PA 15260. E-mail: sreynolds{at}server.ceoh.pitt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clara cell secretory protein (CCSP) is expressed abundantly within the conducting airway epithelium and is thought to have immunoregulatory functions. Differences in the localization of CCSP between mouse and human airways led us to hypothesize that functional homologues of CCSP may compensate for the lack of CCSP expression in proximal airway locations. We previously identified an expressed sequence tag (W82219) whose expression is induced within Clara cells of CCSP knockout mice. Expressed sequence tag W82219 is distantly related to CCSP and represents a member of a new subfamily of secretoglobins (MmSCGB3A2). Another member of the mouse SCGB3 family (MmSCGB3A1) as well as human orthologues (HsSCGB3A1 and HsSCGB3A2) that possess structural homology to CCSP were identified, suggesting they may share common functional properties. SCGB3A1 messenger RNA localizes to a subset of SCGB3A2-expressing cells within bronchi of both mouse and neonatal human lungs. CCSP, SCGB3A1, and SCGB3A2 were decreased in airways of neonates with bronchopulmonary dysplasia and in mice after airway injury. We conclude that secretory cells of the conducting airway epithelium express distinct members of the secretoglobin family in a partially overlapping fashion. Altered expression of secretoglobins in airway disease may contribute to immunoregulatory perturbations commonly seen in chronic airway disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonciliated secretory cells of the conducting airway epithelium represent a heterogeneous population whose phenotype varies dramatically along the proximal to distal airway axis (1–5). This variability in secretory cell character may have a direct influence on the type of proteinaceous secretions contributing to the airway lining fluid at any airway location and may serve an important role in tailoring the composition of airway secretions to specific airway functions both in the steady-state and in airway disease (3). Furthermore, plasticity of secretory cell function plays a critical role in adaptation to acute stresses imposed on the conducting airway such as infection and pollutant exposure (6–11). These findings highlight the importance of a variety of endogenous and exogenous factors in the regulation of secretory cell phenotype and provide insights into mechanisms contributing to pathologic changes to airways observed in chronic airway diseases such as bronchopulmonary dysplasia (BPD), asthma, chronic obstructive pulmonary disease, and cystic fibrosis.

Secretory cells located at distinct airway locations have been classically identified according to morphologic characteristics such as the abundance and type of secretory granules and the presence of agranular endoplasmic reticulum (12). Using these criteria, three broad categories of secretory cells have been defined, including nonciliated bronchiolar (Clara) cells, serous cells, and goblet cells; the latter two populations are present principally in larger airways of the bronchi, trachea, and associated glands (13). However, the more recent application of molecular criteria to define secretory cell subsets has highlighted the potential for functional similarities among morphologically distinct subsets of secretory cells in addition to functional heterogeneity among cells formerly considered to belong to a single category. Expression of Clara cell secretory protein (CCSP, also referred to as Uteroglobin, CC10, CC16, PCB-BP and SCGB1A [14]) exemplifies this disparity between molecular and morphologic definitions of secretory cell subsets. This gene product was first thought of as a molecular marker for Clara cells (15), but it is also detected in both nonciliated bronchiolar (Clara) cells as well as subpopulations of serous and goblet cells within proximal airways of the human lung (16–18). More recently, heterogeneity among CCSP-expressing cells of the mouse lung has been identified as a critical factor allowing for repair of airways after chemically induced ablation of Clara cells (19). In these and other studies, neuroepithelial body-associated CCSP-expressing cells were found to exhibit a naphthalene-resistant phenotype, a functional property that distinguished them from other CCSP-expressing populations in mouse airways (19–21).

Other than CCSP, few molecular markers exist that allow discrimination of secretory cell subsets that do not produce mucin. Alternative murine Clara cell markers include certain CYP450 isoenzymes such as CYP450-2F2. Although CYP450-2F2 is expressed late in lung development relative to CCSP (22), its regional expression in the adult lung is remarkably similar to that of CCSP, the only exception being the lack of CYP450-2F2 expression among neuroepithelial body-associated CCSP-expressing cells (21). Additional Clara cell-specific markers, including tryptase Clara and miniplasmin, have been characterized in rats. However, their expression is common to all nonciliated bronchiolar epithelial cells (23–27). More recently, studies investigating altered gene expression within lungs of CCSP knockout mice demonstrated a compensatory change in the expression of another Clara cell-specific gene, the previously unannotated expressed sequence tag (EST) W82219 (26).

The present study was undertaken using W82219 as a molecular marker to understand heterogeneity among secretory cells of the steady-state and injured mammalian lung. EST W82219, referred to hereon as SCGB3A2, was determined to be a distantly related member of the secretoglobin family, of which CCSP is the founding member (14, 27). CCSP, SCGB3A2, and SCGB3A1 (another closely related family member) were found to define distinct subsets of secretory epithelial cells in conducting airways of the mouse and human lung. As such, these genes provide markers to understand the molecular basis for secretory cell heterogeneity in the normal and developing lung and the response of regionally distinct secretory cell populations to acute and chronic disease.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Database Queries
A search for homologues of MmSCGB3A2 resulted in identification of MmSCGB3A1 as well as distantly related sequences in the EST databanks. These ESTs were sorted into contigs and used to construct consensus nucleotide sequences. These consensus sequences were translated, and the predicted amino acid sequences as well as nucleotide sequences were used to search all databases available for BLAST searching (e.g., nonredundant, ESTs from all organisms, high throughput genomic sequences, Drosophila melanogaster, and Saccharomyces cerevisiae genomes) (28). This process was reiterated with putative new secretoglobin family members. The entire family was aligned with CLUSTAL W (29) and the alignment used to support phylogenetic analyses using the Neighbor Joining/Poisson method. Confirmation of subfamily groupings was performed by bootstrap analysis of the primary sequence (Figure 1) and structural similarities were determined using a variety of structure prediction programs (Meta server: http://BioInfo.PL/meta). Each of the new secretoglobin family members presented in this study was predicted to possess the four helical structures that typify the secretoglobin family (30, 31).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Phylogenetic analysis of the secretoglobin family. Neighbor Joining with Poisson correction was used to generate the tree, after using CLUSTAL W to align the sequences. Lettering above the branches indicates the family designations of the Secretoglobin Nomenclature Committee; we propose 3A for the LuLeu/Hin-1/UGRP branch. Accession numbers for the sequences were as follows: (1) OcCCSP: AAA31497, (2) LcCCSP: AAA30960, (3) MaCCSP: AAL31349, (4) RnCCSP: NP_037183, (5) MmCCSP: NP_035811, (6) SsCCSP: (18), (7) MfCCSP: (18), (8) HsCCSP: NP_003348, (9) MmLGP: AAB67069, (10) McABP: AAD30167, (11) MmdomABP: AAD30166, (12) MmABP: AAB97170, (13) FcFelB: AAC41617, (14) FcFelA: AAC37318, (15)** HsRYD5: BK000201 (maps to telomere of chr 17, clone AF240580), (16) RnRYD5: CAA43068, (17)** MmRYD5: BK000200, (18) MaFHG22: S68231, (19) RnPBPC2: CAA24569, (20)** RnPBPC1BS: BK000198, (21) RnPBPC1: CAA24787, (22) OcLppBL: AAG42804, (23) HsLppA: NP_006543, (24) HsLymphglb: (18), (25) HsLppB: NP_006542, (26)** BtLppAB: nt: BK000199, (EST AI461410), (27) OcLppAS: AAG42803, (28) OcLppAL: AAG42802, (29) MaHGB2: CAB64661, (30) MaHGB1: CAB64660, (31) RnPBPC32: CAB75892, (32) RnPBPC31: AAA41965, (33) OcLPPCL2: AAG42806, (34) OcLppCL: AAG42805, (35) HsMGB1: NP_002402, (36) HsLppC: CAA11865, (37) OcLppCS: AAG42808, (38) OcLppCP: AAG42807, (39) FeldIC2: AAC41616, (40)** MmdIC2Y: BK000197, (41)** MmdIC2C: BK000195, (42)** MmdIC2B: BK000194, (43)** MmdIC2D: BK000196, (44)** MmdIC2A: BK000193, (45) HsLuLeu2/HIN-1 AAK82942, (46)** RnLuLeu2: BK000202 (EST AI011836), (47) MmLuLeu2/HIN-1: AAL26216, (48) MmLuLeu1/UGRP1: AAL25708, (49) HsLuLeu1/UGRP1: AAL26215. **Designates genes submitted to GenBank as part of this study; accession numbers are for nucleotide sequences (that were used for predicting the gene products). All other accession numbers are for amino acid sequences.

 
Animal Housing and Breeding
Adult male FVB/n or 129SvEv wild-type mice (2–4 months of age) were maintained as an in-house colony under specific pathogen-free conditions, and sentinels were tested quarterly using a 16-agent serologic sentinel-screening program. Animals were maintained under a 12-hour light/dark cycle and allowed access to both food and water ad libitum. Timed pregnant females were established by pairing a virgin female with an established male breeder in the late afternoon. The morning on which a vaginal plug was detected was recorded as embryonic Day 0.5. Protocols for the care and use of animals in this study were approved by the IACUC committee of the University of Rochester.

RNA Analysis
All lung RNA was isolated as previously described and included the lobar bronchus (32). Messenger RNA (mRNA) abundance was determined by S1 nuclease protection analysis using previously described methods and templates (33–35). The SCGB3A2 template was a 336-bp HindIII–XhoI fragment of IMAGE clone #403735. Bands intensities were analyzed using a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and were normalized to L32. Results are reported as the mean ± SE. Significance was determined by two-way analysis of variance with challenge (corn oil or naphthalene) and time of recovery (control and 6, 9, 12, 24, 36, and 48 hours) as the two factors analyzed. Significance was accepted at p values less than or equal to 0.05.

Tissue Fixation
Mice were killed by intraperitoneal injection of 100 mg/kg sodium pentobarbital. Adult lung tissue was inflation-fixed with neutral-buffered formalin at 10 cm water pressure for 10 minutes, immersed in neutral-buffered formalin overnight at 4°C, immersed in phosphate-buffered saline overnight at 4°C, dehydrated through graded ethanols, and embedded in paraffin. Embryonic day postcoitus (dpc) 14.5 to 16.5 embryos and dissected lungs from dpc 18.5 and neonates were fixed by immersion in neutral-buffered formalin overnight and processed as detailed for adult mouse tissue.

Human neonatal lung tissue was recovered within 7 hours of death. The right middle lobe, including the main bronchus, was inflation-fixed and processed for histology as described previously. Analysis of CCSP, SCGB3A1, and SCGB3A2 was performed on the tissue block containing the most proximal airways available. Multiple adjacent serial tissue sections from three neonates that died of nonpulmonary complications and four neonates that died of complications associated with BPD were assessed in this study. The representative normal tissue presented in this study was derived from a neonate that was born at 40 weeks gestation, was on mechanical ventilation for less than 24 hours, and died of Pena-Shokeir syndrome at 4 days of age (Subject 98A-168N). The primary disease characteristic of Pena-Shokeir is loss of neuromuscular function that prevents in utero breathing necessary for alveolar development. Arborization and development of the conducting airways is normal in this disease (36). The two other normal tissue samples were derived from infants who were diagnosed with nonketotic hyperglycinemia or hypoxic ischemic encephalopathy/pulmonary hypertension. The representative BPD tissue was derived from a neonate born at 25.4 weeks gestation who died at 65 days of age of BPD with cytomegalo virus infection, after 61 days of mechanical ventilation (Subject 96A-227N). The three other patients with BPD had secondary periventricular leukomalacia or infections at nonpulmonary sites (urinary tract and brainstem). Samples from 19 other neonates with various lung diseases including sepsis/pneumonia, pulmonary hypotension, and unknown etiology were also analyzed, and the results will be reported elsewhere. Protocols for the use of human tissue were approved by the Institutional Review Board of the University of Rochester.

In Situ Hybridization
[35S]-labeled sense and antisense riboprobes (6 x 108 dpm/µg) were generated from CYP450-2F2 complementary DNA (33), MmSCGB3A2 (IMAGE clone #403735), MmSCGB3A1 (IMAGE clone #318938), HsSCGB3A2 (IMAGE clone #3231645), or HsSCGB3A1 (IMAGE clone #1257654) using a Riboprobe Transcription Kit (Promega, Madison, WI). Conditions and solutions for hybridization were as described by Wert and coworkers. (37). Hybridization was performed overnight at 54°C, and slides were washed under high stringency conditions. Slides were dipped in Kodak NTB2 emulsion, exposed for 16 hours, and developed with a Kodak D19 developer, according to the manufacturer's protocol. Images were captured under identical conditions and pseudocolored. In Figures 2–10, silver grains were colored red and superimposed over the brightfield image in Adobe Photoshop.



View larger version (153K):
[in this window]
[in a new window]
  		Figure 2. Localization of MmSCGB3A1 and MmSCGB3A2 in the normal adult murine lung. The spatial distribution of MmSCGB3A2 (A) and MmSCGB3A1 (B) was determined by in situ hybridization (red grains). Images are oriented with the proximal portion of the airway to the right. Hybridization signals were not detected on adjacent sections probed with [35S]-labeled sense riboprobes (data not shown). Original magnification: x10.

 


View larger version (121K):
[in this window]
[in a new window]
  		Figure 10. Localization of CCSP, HsSCGB3A1, and HsSCGB3A2 in the cartilaginous airways and submucosal glands of neonatal human lung compromised by BPD. The spatial distribution of CCSP protein (A, B, and C) was determined by immunohistochemistry, HsSCGB3A2 (D) and HsSCGB3A1 mRNA distribution (E) was determined by in situ hybridization. Thin and thick arrows and arrowheads denote the airway, submucosal glands, and a gland duct, respectively. Original magnification: x400.

 


View larger version (86K):
[in this window]
[in a new window]
  		Figure 3. Localization of MmSCGB3A1 in the normal adult murine trachea. The spatial distribution of MmSCGB3A1 in the superficial epithelium (A, dorsal and B, ventral) and submucosal glands of the trachea (C and D) is shown. Images are oriented with the proximal portion of the airway to the right. Short arrows and asterisks indicate submucosal glands and cartilage rings, respectively. The long arrows in C marks hybridization to the epithelium overlying intercartilaginous regions of the trachea. Original magnification: x100.

 


View larger version (116K):
[in this window]
[in a new window]
  		Figure 4. Localization of CCSP and MmSCGB3A2 during the late pseudoglandular stage of murine lung development. The spatial distribution of CCSP protein (A) and MmSCGB3A2 message (B and C) in lungs of embryonic Day 16.5 mice was determined by immunohistochemistry and in situ hybridization. Analysis of adjacent sagittal sections are presented in A and B, and a transverse section is presented in C. Images are oriented with the dorsal portion of the embryo at the bottom. Thin and thick arrows denote bronchi and bronchioles, respectively. T indicates the developing trachea. Original magnification: x10.

 


View larger version (126K):
[in this window]
[in a new window]
  		Figure 5. Localization of MmSCGB3A1 and MmSCGB3A2 during the early saccular stage of murine lung development. The spatial distribution of MmSCGB3A2 (A) and MmSCGB3A1 (B) in adjacent serial sections of lungs from embryonic Day 18.5 mice is shown. Thin and thick arrows denote bronchi and bronchioles. T and H indicate the developing trachea and heart. Original magnification: x10.

 


View larger version (133K):
[in this window]
[in a new window]
  		Figure 6. Localization of MmSCGB3A2 during the alveolar stage of murine lung development. The spatial distribution of MmSCGB3A1 in the postnatal Day 1 mice is shown. Thin and thick arrows and arrowheads denote bronchi, bronchioles, and terminal bronchioles, respectively. Original magnification: x10.

 


View larger version (104K):
[in this window]
[in a new window]
  		Figure 7. Localization of CCSP, HsSCGB3A1, and HsSCGB3A2 in cartilaginous airways and submucosal glands of the normal neonatal human lung. The spatial distribution of CCSP protein in normal neonatal human lung (A and B) was determined by immunohistochemistry. CCSP immunopositive cells were detected using a rabbit anti-human CCSP antibody and are stained sepia. The tissue is counterstained with hematoxylin. The pattern of HsSCGB3A2 (C and D) and HsSCGB3A1 mRNA expression in normal neonatal human lung (E and F) was determined on adjacent serial sections by in situ hybridization. Boxed regions in A, C, and E are presented at higher magnification in B, D, and F. Thin, thick, and short/thick arrows denote the airway, submucosal glands, and a submucosal gland duct, respectively. Immunoreactivity was not detected on sections analyzed with secondary and tertiary antibodies alone, and hybridization signals were not detected on adjacent sections probed with [35S]-labeled sense riboprobes (data not shown). Original magnification in A, C, and E, x40 and in B, D, and F, x100.

 


View larger version (72K):
[in this window]
[in a new window]
  		Figure 8. Localization of CCSP, HsSCGB3A2, and HsSCGB3A1 in bronchi of the normal neonatal human lung. The spatial distribution of CCSP protein was determined by immunohistochemistry (A) and HsSCGB3A2 (B) and HsSCGB3A1 (C) mRNA by in situ hybridization, as described in the legend to Figure 7. The section in A is adjacent to the serial sections presented in B and C. Original magnification: x100.

 


View larger version (101K):
[in this window]
[in a new window]
  		Figure 9. Localization of CCSP, HsSCGB3A2, and HsSCGB3A1 in terminal bronchioles of the normal neonatal human lung. The spatial distribution of CCSP protein (A) was determined by immunohistochemistry and HsSCGB3A2 (B) and HsSCGB3A1 mRNA distribution (C) was determined by in situ hybridization as described in the legend for Figure 7. Original magnification: x100.

 
Dual In Situ Hybridization and Immunohistochemistry
Tissue was hybridized as described previously with the exception that [3H]-labeled riboprobes were used. After RNase treatment, sections were blocked, then incubated with rabbit anti-rat CCSP (1/16,000) overnight at 4°C. Antigen–antibody complexes were detected as described previously (38) using biotinylated goat anti-rabbit immunoglobulin secondary antibody, streptavidin–horseradish peroxidase, and diamidobenzadine substrate. Slides were then washed sequentially at 65°C for 30 minutes in 50% formamide/2x standard saline citrate/10 mM dithiothreitol, at room temperature for 15 minutes in 2x standard saline citrate/10 mM dithiothreitol, and at room temperature for 15 minutes in 0.1x standard saline citrate/10 mM dithiothreitol. Autoradiography and imaging were performed as described previously.

Immunohistochemistry
Immunohistochemical detection of human CCSP was performed as detailed previously (38) with the exception that rabbit anti-human CCSP (kindly provided by A. Pilon, Claragen, Inc., College Park, MD) was used at a dilution of 1/256,000. This antibody was generated by immunizing rabbits with recombinant human CCSP using standard protocols. This antibody detects a single {bsim} 16 kD protein in human bronchoalveolar lavage (data not shown).

Naphthalene Exposures
FVB/n mice were briefly anesthetized with halothane and injected intraperitoneally with 275 mg naphthalene (dissolved in Mazola corn oil)/kg body weight. Control animals received a comparable dose (10 ml corn oil/kg body weight). Mice were allowed to recover 6, 9, 12, 24, 36, or 48 hours before being killed. Tissues were recovered for RNA and in situ hybridization analysis as described previously.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SCGB3A1 and SCGB3A2 Are Members of the Secretoglobin Family
Previously EST W82219 was shown to be expressed within Clara cells of the normal murine lung and increased in CCSP knockout mice (26). This complementary DNA was used to probe the databases. Multiple homologous ESTs derived primarily from human and mouse lung or lung cancer libraries were identified. Conceptual proteins derived from mouse and human contigs are 91 and 93 amino acids in length, respectively. These proteins are predicted to be hydrophobic (56 and 58% nonpolar residues) and leucine-rich (21 and 23.7%) and were originally termed "mouse" and "human LuLeu1" (39) to highlight their distribution (Lung) and biochemical (Leucine-rich) characteristics. Subsequent BLAST analysis demonstrated that LuLeu1 is identical to the recently identified protein UGRP1, a dexamethasone-responsive gene that is downregulated in T/ebp/Nkx2.1-null mice (40).

Nucleotide and amino acid sequences for mouse and human LuLeu1 were used to query the databanks, resulting in identification of related sequences, which were originally termed "mouse" and "human LuLeu2". The latter proteins are predicted to be small (104 and 106 amino acids), leucine-rich (17.3 and 20.85%), and hydrophobic (63.5 and 60.4% nonpolar residues) and are identical to the previously identified proteins High-In-Normal-1 (HIN-1), a protein that was identified on the basis of decreased expression in breast cancer cells (41) and UGRP2 (40). LuLeu1/UGRP1 and LuLeu2/HIN-1/UGRP2 are members of the secretoglobin family (31, Figure 1) and share a distinct branch of the secretoglobin family tree (Figure 1). In compliance with the convention established by the Secretoglobin Nomenclature Committee (27) LuLeu1/UGRP1 and LuLeu2/HIN-1/UGRP2 will be referred to as SCGB3A2 and SCGB3A1, respectively, and the human and mouse homologues designated Hs (Homo sapiens) and Mm (Mus musculus).

A search for homologues of SCGB3A1 and SCGB3A2 also identified distantly related sequences in the EST databanks, a number of which had not been previously designated to the secretoglobin family. These ESTs were sorted into contigs of consensus sequences, which were used to predict the gene products. Consensus sequences and predicted proteins were used to search all databanks to confirm and extend the secretoglobin family. Putative family members were aligned with CLUSTAL W (29), and the alignment used to support phylogenetic analyses using the Neighbor Joining method with confirmation of subfamily groupings by bootstrap analysis. The most significant addition to the secretoglobin family was made in the SCGB2 branch originally defined by Feline Allergen dI Chain C2. Four full-length and one partial peptide sequence have been added to this subfamily and named mouse allergens dI2A, dI2B, dI2C, dI2D, and dI2Y. Divergence between the feline and murine allergens resulted in subdivision of the SCGB2 branch into the SCGB2A and SCGB2B classes containing the cat and mouse proteins, respectively (Figure 1).

SCGB3A1 Defines a Subset of SCGB3A2-Expressing Cells Within the Conducting Airway Epithelium of Mice
The spatial distribution of SCGB3A1 and SCGB3A2 mRNA was determined by in situ hybridization to adjacent serial sections of adult mouse lung tissue using [35S]-labeled antisense riboprobes. As reported previously, SCGB3A1 mRNA was localized throughout the conducting airway epithelium (Figure 2A, [26]) in a pattern that is similar to that of other Clara cell specific markers including CCSP (20) and CYP450-2F2 (26). Expression of SCGB3A2 is highest in the bronchi and decreases in a shallow gradient along the proximal/distal axis of the airway. In contrast, SCGB3A1 mRNA is expressed predominantly in bronchi with little if any expression in terminal bronchioles (Figure 2B). These data suggest that SCGB3A1 serves as a molecular marker for a subset of airway secretory cells that are enriched in bronchi of mice.

To determine whether SCGB3A1 and SCGB3A2 are expressed in more proximal regions of the murine airway the distribution of these transcripts in the trachea was examined. SCGB3A2 was present at very low levels throughout the tracheal epithelium and associated glands (data not shown). Some variation in relative abundance was found, similar to that observed with SCGB3A1 (see below). SCGB3A1 (Figure 3) was detected in the tracheal epithelium and associated glands and was expressed at higher levels than SCGB3A2 at all locations. Expression of SCGB3A1 message was greater in the ventral tracheal epithelium relative to dorsal (compare Figures 3B and 3A) and increased in a shallow gradient along the proximal to distal axis (compare Figures 3C and 3D with 3B). SCGB3A1 mRNA was detected in epithelial cells lining submucosal glands (Figures 3C and 3D) and was enriched in the surface epithelium overlying intercartilaginous regions of the trachea at some levels (Figures 3C and 3D). In summary, SCGB3A2 is expressed throughout the conducting airway epithelium of the adult murine lung, whereas SCGB3A1 is expressed primarily in the tracheal epithelium, submucosal glands of the proximal trachea, and bronchi.

Regionally Distinct Patterns of SCGB3A1 and SCGB3A2 Gene Expression Are Established During Gestation
To determine whether subsets of secretory cells are established during development, the spatial distribution of SCGB3A1 and SCGB3A2 was determined in the lung at various stages of embryonic development. SCGB3A2 was not detected at dpc 14.5 (data not shown) but was abundantly expressed in both the developing airway (Figures 4B and 4C) and trachea (Figure 4C) by dpc 16.5. Expression of SCGB3A2 preceded that of CCSP at this stage of development (Figure 4A). A similar pattern was observed at dpc 18.5 with continued expression of SCGB3A2 message in the trachea, bronchi, and nascent expression of SCGB3A2 in distal regions of the airway tree (Figure 5A). Between dpc 18.5 (Figure 5A) and postnatal Day 1 (Figure 6), the mature pattern of SCGB3A2 gene expression within the intralobar airway epithelium was established. These data indicate that SCGB3A2 mRNA is expressed throughout the developing airway including the tracheal epithelium and accumulates to detectable levels early in Clara cell differentiation. Consistent with findings in the adult lung, SCGB3A1 mRNA is enriched in the tracheal epithelium at dpc 18.5 with little or no expression detected in the intralobar region of the conducting airway epithelium (Figure 5B) at this time point or at later stages of lung development (Figure 2B and data not shown). These data suggest that the distinct molecular phenotype of secretory cells within the trachea and bronchi are established by dpc 18.5. Moreover, attenuation of SCGB3A1 and SCGB3A2 gene expression in the trachea after birth may mark the separation of distinct lineages of secretory cells in the tracheal and bronchial epithelium.

Regional Expression of SCGB3A1 and SCGB3A2 mRNAs Identify Distinct Secretory Epithelial Cell Subsets Within Human Airway and Submucosal Glands
Significant species differences exist with respect to the abundance, distribution, and phenotypic properties of secretory cells. To determine whether SCGB3A1 and SCGB3A2 serve as useful markers for distinct secretory cell subsets in human airways, the spatial distribution of human SCGB3A1 and SCGB3A2 mRNA in normal neonatal human lung tissue was investigated. Tissue from three neonates that died of nonpulmonary complications was analyzed, and data from one representative individual is presented. Human SCGB3A2, like the mouse homologue, is expressed throughout the conducting airway epithelium of the neonatal lung in a pattern similar to that of human CCSP. SCGB3A2 mRNA levels were slightly higher in large caliber cartilaginous airways (Figures 7C and 7D) than in midlevel bronchus (Figure 8B) or in bronchioles (Figure 9B). The number of SCGB3A2-expressing cells was similar to that of CCSP-expressing cells at all airway locations (Figures 7A, 7B, 8A, and 9A), and neither mRNA was detected in respiratory bronchioles, alveolar ducts, or alveolar epithelium (Figures 7–9).

The distribution of human SCGB3A1 mirrored that of mouse SCGB3A1 and was expressed predominantly in large airways (Figures 7E and 7F) and midlevel bronchus (Figure 8C). Little HsSCGB3A1 was detected in bronchioles (Figure 9C). Adjacent section analysis of human SCGB3A1 and SCGB3A2 expression patterns in human submucosal gland further supports the notion that these gene products define subsets of secretory cells. Human SCGB3A2 is expressed primarily in serous-like cells of the submucosal gland acinus (Figure 7D). In contrast, SCGB3A1 is expressed at relatively high levels in both acinar and ductile cells of the submucosal gland (Figure 7F). This pattern is similar to that of human CCSP protein-expressing cells (Figure 7B). These data are consistent with findings in mouse airways that indicate SCGB3A1 and SCGB3A2 serve as molecular markers that identify distinct subsets of secretory cells within the surface and glandular epithelia of conducting airways.

SCGB3A1 and SCGB3A2 Gene Expression Is Altered in BPD
Involvement of secretory cells in the etiology of various human lung diseases has been implicated by alterations in the concentration of CCSP in bronchoalveolar lavage (42–46) and in the number of CCSP-expressing cells (18, 47, 48). SCGB3A1 and SCGB3A2 gene expression was investigated in lung tissue from human neonates that died of complications associated with BPD. Tissues from four neonates that died of BPD were analyzed, and representative data from one individual is presented. Squamous metaplasia of the epithelium and decreased epithelial density was particularly apparent in bronchi with minor changes to the epithelium of submucosal glands (Figure 10). In contrast with normal tissue, little or no CCSP protein (Figures 10A–10C) or SCGB3A2 and SCGB3A1 message (Figures 10D and 10E) was detected in the upper airway epithelium. The abundance and distribution of CCSP (Figures 10A–10C) and SCGB3A2 (Figure 10D) and SCGB3A1 (Figure 10E) mRNA in submucosal glands and ducts were similar to normal controls (Figures 7C–7F). Small caliber airways including terminal bronchioles were absent or severely attenuated in three of the four patients with BPD who were studied, and residual cells did not express CCSP, SCGB3A1, or SCGB3A2 (data not shown). Although this study used a relatively small sample population these results suggest that injury to and remodeling of the airway epithelium, particularly secretory cells that express CCSP, SCGB3A1, and SCGB3A2, are a pathologic feature of BPD. It remains to be determined what functional significance this may have with respect to either resolution or further exacerbation of lung disease.

SCGB3A2 Is a Sensitive Molecular Marker for Clara Cells
We have previously demonstrated that SCGB3A1 is expressed within Clara cells of the steady-state airway epithelium and have suggested that this message serves as a molecular marker for the Clara cell lineage (26). To test this hypothesis the parenteral naphthalene exposure model was employed. Napthalene exposure results in greater than 90% depletion of Clara cells within 2 days and is followed by a period of rapid Clara cell regeneration involving activation of airway stem cells (20, 21). The utility of SCGB3A2 as an early Clara/secretory cell marker was further tested by comparison of changes in the abundance of its mRNA with that CYP450-2F2. Male FVB/n mice were exposed to 275 mg/kg naphthalene (intraperitoneally) and killed after 6-, 9-, 12-, 24-, 36-, and 48-hour recovery periods. mRNA abundance was assessed by S1 nuclease protection analysis and normalized to L32. Parallel decreases in the abundance of CYP450-2F2 and SCGB3A2 mRNAs occurred 6 to 48 hours after naphthalene exposure (Figure 11A). The abundance of SCGB3A2 message declines to its lowest level 48 hours after exposure (16 and 10% of control mice, respectively) and was significantly different from control at 9, 12, 36, and 48 hours after exposure.



View larger version (79K):
[in this window]
[in a new window]
  		Figure 11. SCGB3A2 and CyP450-2F2 expression after naphthalene-mediated Clara cell depletion. Adult male FVB/n mice (four animals/group) were treated with 375 mg naphthalene/mg body weight, allowed to recover 6 to 48 hours, and total lung RNA recovered. Abundance of SCGB3A2 and CYP450-2F2 was determined by S1 nuclease protection analysis (A). Values for CYP450-2F2 (white bars) and SCGB3A2 (black bars) were quantified using a phosphoimager, normalized to L32, and the mean ± SE reported. Asterisks indicate values that are significantly different from untreated control mice. The distribution of MmSCGB3A2 transcript and CCSP protein was determined 72 hours after naphthalene exposure by dual in situ hybridization using [3H]-labeled MmSCGB3A2 antisense riboprobes and CCSP immunohistochemistry (B). Autoradiographic grains are shown in black and immunoreactivity is shown in sepia. Arrows indicate nascent SCGB3A2-expression cells at the leading edge of the regenerative domain.

 
The pattern of SCGB3A2 gene expression in the repairing epithelium of male FVB/n mice 72 hours after exposure to 275 mg/kg naphthalene was determined by in situ hybridization using a [3H]-labeled antisense riboprobe. In contrast with the normal epithelium (Figure 2A) SCGB3A2 mRNA was restricted to focal regions of the regenerating bronchiolar epithelium (Figure 11B), particularly branchpoints, the predominant location of nascent Clara cells (24, 25). SCGB3A2 message was coexpressed with CCSP protein at branchpoints (Figure 11B), indicating that SCGB3A2 message was expressed in nascent Clara cells during regeneration of the injured airway epithelium (Figure 11B). In contrast, a subset of SCGB3A1 mRNA-expressing cells located at the leading edge of the regenerative domain (Figure 11B, arrows) do not contain detectable levels of CCSP protein, a result that most likely reflects differences in the kinetics of message and protein accumulation. Thus, SCGB3A2 is expressed in nascent Clara cells and serves as an early differentiation marker for the Clara cell lineage. These findings suggest that SCGB3A2 is a sensitive measure of Clara cell depletion and regeneration after naphthalene-mediated Clara cell injury and that this gene product may be useful as a biomarker for Clara cell function in health and disease.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that two members of the secretoglobin gene family, SCGB3A1 and SCGB3A2, define molecularly distinct subsets of secretory cells within the conducting airway epithelium. Analysis of SCGB3A2 gene expression in the murine lung indicates that this gene product is an early molecular marker for Clara cells (CCSP-expressing nonciliated secretory cells distributed throughout the conducting airway epithelium [49]). In contrast with CCSP and SCGB3A2, SCGB3A1 is highly expressed in a subset of airway epithelial cells located primarily in the bronchi. The spatial distribution of SCGB3A1 and SCGB3A2 in human neonatal lung is similar to that observed in mice with the exception that these genes are more highly expressed in human bronchi and associated glands. Thus, these genes provide tools for analysis of the molecular mechanisms underlying establishment and maintenance of secretory cell subpopulations in the normal lung as well as phenotypic transitions associated with lung disease.

Mechanisms governing the establishment of a functionally diverse secretory epithelium have yet to be established, although available data suggest a progressive restriction of cell fate. In the mouse, secretory cell differentiation begins during the early pseudoglandular period and is marked by expression of CCSP in the bronchi (37). During the ensuing 24 hours of development (late pseudoglandular period) the domain of CCSP-expressing cells expands to include the entire primordial airway as well as the trachea. Expression of SCGB3A2 recapitulates this pattern with the exception that expression of this gene in the trachea precedes that of CCSP. Although the initial phase of SCGB3A2 gene expression in the tracheal epithelium may be indicative of a period of promiscuous gene expression analogous to that described in the intrapulmonary airways (37, 50), robust expression of this marker suggests that secretory cells of the trachea and bronchi are specified simultaneously rather than sequentially. Therefore, SCGB3A2, like CCSP, is a very early secretory cell marker, which strongly suggests considerable overlap in the transcription factors that both control their expression and specify cell lineage.

The existence of a secondary developmental program that subdivides the airway into distinct upper and lower regions is suggested by restricted expression of SCGB3A1 in the trachea and bronchi. Subsequent downregulation of SCGB3A1 and SCGB3A2 gene expression in the murine tracheal epithelium during the postnatal period may mark a third phase layer of regional specialization that distinguishes the tracheal and bronchial epithelium of the mouse. Identification of factors that control spatially restricted expression of SCGB3A1 and downregulation of SCGB3A1 and SCGB3A2 in the murine tracheal epithelium may lead to further insights into cellular and cell/matrix interactions leading to regional specialization of the airway epithelium.

Functional specialization of nonciliated secretory cells within the mature lung is suggested by ultrastructural and biochemical distinctions between cells of the bronchi and bronchioles (51). Within the murine airway, bronchial Clara cells exhibit a columnar-to-low cuboidal morphology and have relatively more granular endoplasmic reticulum and less smooth endoplasmic reticulum than their tall columnar counterparts in the bronchioles (51). Differences in CYP450 enzymatic activity (52, 53), cellular glutathione pool size (54–57), and glutathione regenerative capacity (55, 56) further distinguish these cells and may contribute variation in pollutant sensitivity (22, 56–58). Regionally specialized secretory activity is suggested by variation in the number, density, and morphologic characteristics of secretory granules and granular endoplasmic reticulum (6, 12, 51, 59), differences in cytoplasmic and granular CCSP concentration (6, 60), and in resynthesis of CCSP after induced secretion (12). Results of the present study indicate that secretory cells within the bronchi of the murine lung are further distinguished from bronchiolar secretory cells by their expression of SCGB3A1 message, which implicates differences in their repertoire of secreted proteins. The potential for functional homology among ultrastructurally distinct secretory cells of human and murine bronchi is suggested by similarities in the distribution of SCGB3A1 in these species. This and other differentially expressed proteins are likely determinants of the unique biophysical and biochemical characteristics of the extracellular lining fluid that bathes the upper airway and may contribute to protection of this region from environmental and infectious agents.

Expression of CCSP and SCGB3A2 by secretory cells distributed throughout the mature airway highlights the molecular continuity of various nonciliated secretory cell populations despite distinct morphologic and functional characteristics. Results of this study indicate that the gene expression profile of secretory cells within neonatal human submucosal glands, which includes CCSP as well as SCGB3A1 and SCGB3A2, overlaps with that of secretory cells of the bronchi. Expression of CCSP in submucosal glands has not been reported previously, and the functional implication of altered CCSP gene expression in mucosecretory diseases involving submucosal gland pathology requires further investigation. In the lower airway of the neonatal human and murine lung, secretory cells with distinct morphologic features share a common gene expression profile defined by CCSP and SCGB3A2. These data suggest that functionally analogous secretory cells within the morphologically diverse human and mouse airway can be defined by expression of secretoglobin gene family members.

A role for altered Clara cell function in the etiology of chronic airway disease is indicated by decrements in the number of CCSP immunopositive cells (47, 48) and alterations in CCSP concentration in the bronchoalveolar lavage fluid of individuals with asthma (48), chronic obstructive pulmonary disease (61), and BPD (42–45). These observations have led to the suggestion that CCSP exerts a direct effect on pulmonary immunoregulation. Our studies in CCSP null mice demonstrate that altered CCSP gene expression is associated with compensatory increases in the expression of both SCGB3A2 (26) and SCGB3A1 mRNAs (S.D.R. and B.R.S., unpublished observation), in addition to altered expression of immunoglobulin A (26). Thus, CCSP as well as other secretoglobins may exert regulatory influences on the inflammatory response. CCSP has been shown to regulate neutrophil migration (62–64) in vitro and inhibits the production and biologic activity of interferon {gamma} (65). Insights into possible mechanisms mediating these antiinflammatory activities of CCSP have not been completely elucidated. However, the finding that CCSP-/- mice show identical responses to inhaled bacterial endotoxin (66), yet exhibit differences in hyperoxia-induced pulmonary inflammation (67), raises the possibility that antiinflammatory effects of CCSP may specifically affect the CD18-independent pathway of neutrophil emigration (68).

We have demonstrated that BPD is associated with quantitative differences in expression of CCSP, SCGB3A1, and SCGB3A2 in the surface epithelium of the upper and lower airway. This complex disease, which reflects the response of the developing lung to barotrauma and oxidant injury, is generally associated with immaturity of the parenchyma and characterized by alveolar simplification and decreased alveolar number and surface area. However, this disease also affects the airway and is characterized by squamous metaplasia and scarring of both central and peripheral airway, increased airway smooth muscle, and peribronchiolar fibrosis (69). The finding that airway changes are observed both in neonates with early BPD and late-stage BPD suggests that this pathology is established during initiation of the disease process. Interestingly, patients with BPD are at very high risk for development of pneumonia, especially viral infection. Our finding that airway cell phenotype is altered in patients with or without CMV suggests that viral infection is not the primary determinant of airway pathology in BPD. Further studies using a larger sample size are necessary to determine whether the decreased expression of CCSP, SCGB3A1, and SCGB3A2 that are associated with BPD contribute to elevated susceptibility to lower respiratory tract infections.

The secretoglobin family is defined by similarities in the genomic structure of individual genes and in the protein structures, whereas subgroups are based on differences in the amino acid sequences. The genomic structure of secretoglobin family members is remarkably conserved, consisting of three exons in which the first splice junction is within the amino acid 18 codon, the first intron is 1 to2 kb, the second exon is spliced to leave 8 to 9 amino acids in the third exon, and the second intron is significantly shorter than the first intron, usually ca. 300 nt. Secretoglobin proteins, which are expressed in epithelial secretory tissues and appear to be found only in mammals, have a highly conserved signal sequence of 20 amino acids and a mature domain of 70 to 80 amino acids. With some variation, the mature domains of the proteins contain cysteine residues at or near the N- and C-termini, and these cysteines form disulfide bridges to create an antiparallel dimer. Studies of the CCSP crystal structure demonstrate that the each monomer forms four {alpha} helices, three of which are aligned to form a hydrophobic pocket (30). Small hydrophobic ligands are coordinated by a tyrosine within this region of the CCSP homodimer and may contribute to cellular activities via receptor-mediated signaling (70). The predicted ternary structures for other secretoglobin proteins indicate that the four helical coil structures are highly conserved within this family, although amino acid identities among the individual family members may range lower than 10% (30, this study).

Structural relationships among members of the secretoglobin gene family suggest functional similarities that may be associated with binding of small hydrophobic ligands. Although CCSP is the most extensively analyzed of the secretoglobins, other members of this large gene family may function in the regulation of the local immune response within the lung (SCGB3A2/UGRP1 and SCGB3A1/UGRP2; 40, 71) or in other secretory epithelia including the prostate (prostatic steroid binding protein; 72), mammary gland (SCGB3A1/HIN-1, 41), and lacrimal gland (73). Parallels in hormonal regulation of SCGB3A1, SCGB3A2, and mammoglobin gene expression and in immunomodulation by CCSP, prostatic steroid binding protein, and the lipophilins suggest functional continuity among secretoglobin gene family members in secretory epithelia of mammals. Further investigation into contributions made by SCGB3 (i.e., SCGB3A1 and SCGB3A2) and SCGBA1 (i.e., CCSP) secretoglobins in regional regulation of inflammatory and immunologic responses may yield critical new insights into mechanisms of airway dysfunction that accompany chronic lung disease.

NOTE IN PROOF:
The nomenclature used to designate SCGB3A1 and SCGB3A2 was altered in the current manuscript to reflect that established by Porter and colleagues (Mechanisms of Development 2002;114:201–204).


   Acknowledgments
 
The authors are grateful to Aprile Pilon of Claragen, Inc. who kindly provided antiserum specific for human CCSP; Paul D. Kingsley for assistance in imaging; Adam Giangreco for naphthalene time course RNA; Kyung U. Hong for tracheal tissue; and Cheryl Hurley, Heidie Huyck, and Candace Hrelec for technical assistance

Accession Numbers

MmdIC2A: BK000193

MmdIC2B: BK000194

MmdIC2C: BK000195

MmdIC2D: BK000196

MmdIC2Y: BK000197

RnPBPC1BS: BK000198

BtLppAB: BK000199

MmRYD5: BK000200

HsRYD5: BK000201

RnLuLeu2/HIN-1: BK000202


   FOOTNOTES
 
This work was supported by research grants: HL64888, ES08964, and HL70575.

Received in original form April 4, 2002; accepted in final form July 25, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Plopper CG, Halsebo JE, Berger WJ, Sonstegard KS, Nettesheim P. Distribution of nonciliated bronchiolar epithelial (Clara) cells in intra- and extrapulmonary airways of the rabbit. Exp Lung Res 1983;5:79–98.[Medline]
  2. Pack RJ, Al-Ugaily LH, Morris G. The cells of the tracheobronchial epithelium of the mouse: a quantitative light and electron microscope study. J Anat 1981;132:71–84.[Medline]
  3. Massaro GD, Singh G, Mason R, Plopper CG, Malkinson AM, Gail DB. Biology of the Clara cell. Am J Physiol 1994;266:L101–L106.[Free Full Text]
  4. Tomkiewicz RP, Albers GM, De Sanctis GT, Ramirez OE, King M, Rubin BK. Species differences in the physical and transport properties of airway secretions. Can J Physiol Pharmacol 1995;73:165–171.[Medline]
  5. Jiang Q, Engelhardt JF. Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis. Eur J Hum Genet 1998;6:12–31.[CrossRef][Medline]
  6. Dodge DE, Rucker RB, Singh G, Plopper CG. Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy. J Histochem Cytochem 1993;41:1171–1183.[Abstract]
  7. Pinkerton KE, Dodge DE, Cederdahl-Demmler J, Wong VJ, Peake J, Haselton CJ, Mellick PW, Singh G, Plopper CG. Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months. Am J Pathol 1993;142:1–10.
  8. Plopper CG, Duan X, Buckpitt AR, Pinkerton KE. Dose-dependent tolerance to ozone. IV: site-specific elevation in antioxidant enzymes in the lungs of rats exposed for 90 days or 20 months. Toxicol Appl Pharmacol 1994;127:124–131.[CrossRef][Medline]
  9. Harkema JR, Hotchkiss JA. Ozone- and endotoxin-induced mucous cell metaplasias in rat airway epithelium: novel animal models to study toxicant-induced epithelial transformation in airways. Toxicol Lett 1993;68:251–263.[CrossRef][Medline]
  10. Vernooy JH, Dentener MA, van Suylen RJ, Buurman WA, Wouters EF. Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology. Am J Respir Cell Mol Biol 2002;26:152–159.[Abstract/Free Full Text]
  11. Yanagihara K, Seki M, Cheng PW. Lipopolysaccharide induces mucus cell metaplasia in mouse lung. Am J Respir Cell Mol Biol 2001;24:66–73.[Abstract/Free Full Text]
  12. Dodge DE, Plopper CG, Rucker RB. Regulation of Clara cell 10 kD protein secretion by pilocarpine: quantitative comparison of nonciliated cells in rat bronchi and bronchioles based on laser scanning confocal microscopy. Am J Respir Cell Mol Biol 1994;10:259–270.[Abstract]
  13. Basbaum CB, Finkbeiner WE. Airway secretion: a cell-specific analysis. Horm Metab Res 1988;20:661–667.[Medline]
  14. Ni J, Kalff-Suske M, Gentz R, Schageman J, Beato M, Klug J. All human genes of the uteroglobin family are localized on chromosome 11q12.2 and form a dense cluster. Ann NY Acad Sci 2000;923:25–42.[Abstract/Free Full Text]
  15. Singh G, Katyal SL. Clara cells and Clara cell 10 kD protein (CC10). Am J Respir Cell Mol Biol 1997;17:141–143.[Free Full Text]
  16. Engelhardt JF, Zepeda M, Cohn JA, Yankaskas JR, Wilson JM. Expression of the cystic fibrosis gene in adult human lung. J Clin Invest 1994;93:737–749.
  17. Hay JG, Danel C, Chu CS, Crystal RG. Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease. Am J Physiol 1995;268:L565–L575.[Abstract/Free Full Text]
  18. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of Clara cells in normal human airway epithelium. Am J Respir Crit Care Med 1999;159:1585–1591.[Abstract/Free Full Text]
  19. Hong KU, Reynolds SD, Giangreco A, Hurley CM, Stripp BR. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol 2001;24:671–681.[Abstract/Free Full Text]
  20. Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269:L791–L799.[Abstract/Free Full Text]
  21. Reynolds SD, Giangreco A, Power JH, Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol 2000;156:269–278.[Abstract/Free Full Text]
  22. Fanucchi MV, Murphy ME, Buckpitt AR, Philpot RM, Plopper CG. Pulmonary cytochrome P450 monooxygenase and Clara cell differentiation in mice. Am J Respir Cell Mol Biol 1997;17:302–314.[Abstract/Free Full Text]
  23. Kido H, Yokogoshi Y, Sakai K, Tashiro M, Kishino Y, Fukutomi A, Katunuma N. Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells: a possible activator of the viral fusion glycoprotein. J Biol Chem 1992;267:13573–13579.[Abstract/Free Full Text]
  24. Sakai K, Kawaguchi Y, Kishino Y, Kido H. Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and influenza virus. J Histochem Cytochem 1993;4:89–93.
  25. Murakami M, Towatari T, Ohuchi M, Shiota M, Akao M, Okumura Y, Parry MA, Kido H. Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus. Eur J Biochem 2001;268:2847–2855.[Medline]
  26. Watson TM, Reynolds SD, Mango GW, Boe IM, Lund J, Stripp BR. Altered lung gene expression in CCSP-null mice suggests immunoregulatory roles for Clara cells. Am J Physiol 2001;281:L1523–L1530.[Abstract/Free Full Text]
  27. Klug J, Beier HM, Bernard A, Chilton BS, Fleming TP, Lehrer RI, Miele L, Pattabiraman N, Singh G. Uteroglobin/Clara cell 10-kDa family of proteins: nomenclature committee report. Ann NY Acad Sci 2000;923:348–354.[Free Full Text]
  28. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–3402.[Abstract/Free Full Text]
  29. Thompson JD, Higgins DG, Gibson TJ. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–4680.[Abstract/Free Full Text]
  30. Callebaut I, Poupon A, Bally R, Demaret J-P, Housset D, Delettre J, Hossenlopp P, Mornon J-P. The uteroglobin fold. Ann N Y Acad Sci 2000;923:90–112.[Abstract/Free Full Text]
  31. Pattabiraman N, Matthews JH, Ward KB, Mantile-Selvaggi G, Miele L, Mukherjee AB. Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding. Ann NY Acad Sci 2000;923:113–127.[Abstract/Free Full Text]
  32. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.[Medline]
  33. Stripp BR, Lund J, Mango GW, Doyen KC, Johnston C, Hultenby K, Nord M, Whitsett JA. Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am J Physiol 1996;271:L656–L664.[Abstract/Free Full Text]
  34. Mango GW, Johnston CJ, Reynolds SD, Finkelstein JN, Plopper CG, Stripp BR. Clara cell secretory protein deficiency increases oxidant stress response in conducting airways. Am J Physiol 1998;275:L348–L356.[Abstract/Free Full Text]
  35. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  36. Itoh H, Chuganji Y, Kodama Y, Seguchi T, Kataoka H, Ikenoue T, Koono M. Pena-shokeir type I syndrome with thymic and systemic lymphoid hyperplasia: report of an autopsy case. Hum Pathol 2000;31:1321–1324.[CrossRef][Medline]
  37. Wert SE, Glasser SW, Korfhagen TR, Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 1993;156:426–443.[CrossRef][Medline]
  38. Reynolds SD, Mango GW, Gelein R, Boe IM, Lund J, Stripp BR. Normal function and lack of fibronectin accumulation in kidneys of Clara cell secretory protein/uteroglobin deficient mice. Am J Kidney Dis 1999;33:541–551.[Medline]
  39. Reynolds SD, Watson TM, Stripp BR. LuLeu, a lung-specific leucine rich product of airway epithelial cells that is enriched within early Clara cell progenitors [abstract]. Am J Respir Crit Care Med 2001;163:A468.
  40. Niimi T, Keck-Waggoner CL, Popescu NC, Zhou Y, Levitt RC, Kimura S. UGRP1, a uteroglobin/Clara cell secretory protein-related protein, is a novel lung-enriched downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Mol Endocrinol 2001;15:2021–2036.[Abstract/Free Full Text]
  41. Krop IE, Sgroi D, Porter DA, Lunetta KL, LeVangie R, Seth P, Kaelin CM, Rhei E, Bosenberg M, Schnitt S, et al. HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells. Proc Natl Acad Sci USA 2001;98:9796–9801.[Abstract/Free Full Text]
  42. Anderson O, Noack G, Robertson B, Glaumann H, Sonnenfeld T, Lund J. Ontogeny of a human polychlorinated biphenyl-binding protein: level of expression in tracheal aspirates in bronchopulmonary dysplasia. Chest 1994;105:17–22.[Abstract/Free Full Text]
  43. Khoor A, Gray ME, Singh G, Stahlman MT. Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia. J Histochem Cytochem 1996;44:1429–1438.[Abstract]
  44. Pilon AL. Rationale for the development of recombinant human CC10 as a therapeutic for inflammatory and fibrotic disease. Ann NY Acad Sci 2000;923:280–299.[Abstract/Free Full Text]
  45. Ramsay PL, DeMayo FJ, Hegemier SE, Wearden ME, Smith CV, Welty SE. Clara cell secretory protein oxidation and expression in premature infants who develop bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001;164:155–161.[Abstract/Free Full Text]
  46. Broeckaert F, Bernard A. Clara cell secretory protein (CC16): characteristics and perspectives as lung peripheral biomarker. Clin Exp Allergy 2000;30:469–475.[CrossRef][Medline]
  47. Shijubo N, Itoh Y, Yamaguchi T, Imada A, Hirasawa M, Yamada T, Kawai T, Abe S. Clara cell protein-positive epithelial cells are reduced in small airways of asthmatics. Am J Respir Crit Care Med 1999;160:930–933.[Abstract/Free Full Text]
  48. Shijubo N, Itoh Y, Yamaguchi T, Sugaya F, Hirasawa M, Yamada T, Kawai T, Abe S. Serum levels of Clara cell 10-kDa protein are decreased in patients with asthma. Lung 1999;177:45–52.[CrossRef][Medline]
  49. Clara M. Zur Histobiologie des Bronchialepithels. Z Mikrosk-Anat Forsch 1937;41:321–347.
  50. Wuenschell CW, Sunday ME, Singh G, Minoo P, Slavkin HC, Warburton D. Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages. J Histochem Cytochem 1996;44:113–123.[Abstract]
  51. Plopper CG. Comparative morphologic features of bronchiolar epithelial cells: the Clara cell. Am Rev Respir Dis 1983;128:S37–S41.[Medline]
  52. Philpot RM. Modulation of the pulmonary cytochrome P450 system as a factor in pulmonary-selective toxic responses: fact and fiction. Am J Respir Cell Mol Biol 1993;9:347–349.
  53. Forkert PG. 1,1-Dichloroethylene-induced Clara cell damage is associated with in situ formation of the reactive epoxide: immunohistochemical detection of its glutathione conjugate. Am J Respir Cell Mol Biol 1999;20:1310–1318.[Abstract/Free Full Text]
  54. Duan X, Buckpitt AR, Pinkerton KE, Ji C, Plopper CG. Ozone-induced alterations in glutathione in lung subcompartments of rats and monkeys. Am J Respir Cell Mol Biol 1996;14:70–75.[Abstract]
  55. West JA, Buckpitt AR, Plopper CG. Elevated airway GSH resynthesis confers protection to Clara cells from naphthalene injury in mice made tolerant by repeated exposures. J Pharmcol Exp Ther 2000;294:516–523.[Abstract/Free Full Text]
  56. West JA, Chichester CH, Buckpitt AR, Tyler NK, Brennan P, Helton C, Plopper CG. Heterogeneity of Clara cell glutathione: a possible basis for differences in cellular responses to pulmonary cytotoxicants. Am J Respir Cell Mol Biol 2000;23:27–36.[Abstract/Free Full Text]
  57. Plopper CG, Van Winkle LS, Fanucchi MV, Malburg SR, Nishio SJ, Chang A, Buckpitt AR. Early events in naphthalene-induced acute Clara cell toxicity. II: comparison of glutathione depletion and histopathology by airway location. Am J Respir Cell Mol Biol 2001;24:272–281.[Abstract/Free Full Text]
  58. Van Winkle LS, Buckpitt AR, Plopper CG. Maintenance of differentiated murine Clara cells in microdissected airway cultures. Am J Respir Cell Mol Biol 1996;14:586–598.[Abstract]
  59. Wasano K, Hirakawa Y. Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study. Histochemistry 1992;98:165–171.[CrossRef][Medline]
  60. Bedetti CD, Singh J, Singh G, Katyal SL, Wong-Chong ML. Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies. J Histochem Cytochem 1987;35:789–794.[Abstract]
  61. Pilette C, Godding V, Kiss R, Delos M, Verbeken E, Decaestecker C, De Paepe K, Vaerman JP, Decramer M, Sibille Y. Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;163:185–194.[Abstract/Free Full Text]
  62. Mukherjee AB, Cordella-Miele E, Kikukawa T, Miele L. Modulation of cellular response to antigens by uteroglobin and transglutaminase. Adv Exp Med Biol 1988;231:135–152.[Medline]
  63. Miele L, Cordella-Miele E, Facchiano A, Mukherjee AB. Inhibition of phospholipase A2 by uteroglobin and antiflammin peptides. Adv Exp Med Biol 1990;279:137–160.[Medline]
  64. Miele L, Cordella-Miele E, Mantile G, Peri A, Mukherjee AB. Uteroglobin and uteroglobin-like proteins: the uteroglobin family of proteins. J Endocrinol Invest 1994;17:679–692.[Medline]
  65. Magdaleno SM, Wang G, Jackson KJ, Ray MK, Welty S, Costa RH, DeMayo FJ. Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro. Am J Physiol 1997;272:L1142–L1151.[Abstract/Free Full Text]
  66. Johnston CJ, Finkelstein JN, Oberdorster G, Reynolds SD, Stripp BR. Clara cell secretory protein-deficient mice differ from wild-type mice in inflammatory chemokine expression to oxygen and ozone, but not to endotoxin. Exp Lung Res 1999;25:7–21.[CrossRef][Medline]
  67. Johnston CJ, Mango GW, Finkelstein JN, Stripp BR. Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice. Am J Respir Cell Mol Biol 1997;17:147–155.[Abstract/Free Full Text]
  68. Doerschuk CM, Tasaka S, Wang Q. CD11/CD18-dependent and -independent neutrophil emigration in the lungs: how do neutrophils know which route to take? Am J Respir Cell Mol Biol 2000;23:133–136.[Free Full Text]
  69. Margraf LR, Tomashefski JF, Bruce MC, Dahms BB. Morphometric analysis of the lung in bronchopulmonary dysplasia. Am Rev Respir Dis 1991;143:391–400.[Medline]
  70. Burmeister R, Boe IM, Nykjaer A, Jacobsen C, Moestrup SK, Verroust P, Christensen EI, Lund J, Willnow TE. A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney. J Biol Chem 2001;276:13295–13301.[Abstract/Free Full Text]
  71. Niimi T, Munakata M, Keck-Waggoner CL, Popescu NC, Levitt RC, Hisada M, Kimura S. A polymorphism in the human UGRP1 gene promoter that regulates transcription is associated with an increased risk of asthma. Am J Hum Genet 2002;70:718–725.[CrossRef][Medline]
  72. Maccioni M, Riera CM, Rivero VE. Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor. J Reprod Immunol 2001;50:133–149.[CrossRef][Medline]
  73. Lehrer RI, Nguyen T, Zhao C, Ha CX, Glasgow BJ. Secretory lipophilins: a tale of two species. Ann NY Acad Sci 2000;923:59–67.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
A. J. Ross, L. A. Dailey, L. E. Brighton, and R. B. Devlin
Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells
Am. J. Respir. Cell Mol. Biol., August 1, 2007; 37(2): 169 - 185.
[Abstract] [Full Text] [PDF]